Influenza antigen delivery vectors and constructs

ABSTRACT

The present invention relates to fluorocarbon vectors for the delivery of influenza antigens to immunoresponsive target cells. It further relates to fluorocarbon vector-influenza antigen constructs and the use of such vectors associated with antigens as vaccines and immunotherapeutics in animals, including humans.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/139,293 filed Dec. 23, 2013, which is a continuation applicationof U.S. application Ser. No. 12/201,894, filed Aug. 29, 2008, now U.S.Pat. No. 8,642,531, which claims the benefit of and priority to U.S.Provisional Patent Application Ser. No. 60/969,481 filed Aug. 31, 2007.This application also claims the benefit of and priority to GreatBritain Patent Application Serial No. GB0716992.3 filed Aug. 31, 2007.The entire disclosure of each application is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to fluorocarbon vectors for the deliveryof influenza antigens to immunoresponsive target cells. It furtherrelates to fluorocarbon vector-influenza antigen constructs and the useof such vectors associated with antigens as vaccines andimmunotherapeutics in animals, including humans.

BACKGROUND

Influenza is the generic term for diseases or infections caused by theinfluenza virus. Influenza viruses are members of the Orthomyxoviridaefamily of viruses and comprise two genera: influenza A and B viruses,and influenza C virus. Influenza A, B and C viruses are distinguished onthe basis of their internal nucleoprotein and matrix proteins which arespecific for each viral type. Influenza A viruses are naturally able toinfect a range of animal species, including humans, swine, birds, sealsand horses. Influenza B viruses, however, infect only humans, whilstinfluenza C virus infects humans and swine. Influenza A viruses arefurther categorised into subtypes that are determined by theantigenicity of the surface glycoproteins, the haemagglutinin (H) andneuraminidase (N).

Historically, influenza A human infections have been caused by threesubtypes of haemagglutinin (H1, H2 and H3) and two neuraminidasesubtypes (N1 and N2); more recently human infections by the previouslyavian-restricted subtypes H5, H7 and H9 have also been reported. A totalof 16 distinct haemagglutinin and 9 neuraminidase influenza A subtypeshave been identified to date; these are all prevalent in birds. Swineand horses, like humans, are limited to a much narrower range ofsubtypes.

Influenza A and B virions are pleiomorphic in structure, sphericalexamples being 80-120 nm in diameter, whilst filamentous forms may be upto 300 nm in length. There are approximately 500 surface spikeglycoproteins per particle (usually in the ratio of four to fivehaemagglutinin proteins to one neuraminidase) that are embedded in ahost-derived lipid bilayer membrane. Within the membrane is thetransmembrane ion channel protein M2, whilst the structural protein M1underlies the bilayer. Within the core of the virus, the single strandednegative sense RNA is associated with the six other viral proteinsexpressed from its genome: the nucleoprotein (NP), three transcriptases(PB2, PB1, and PA) and two nonstructural proteins (NS1 and NS2). Theinfluenza virus genome comprises eight segments; a feature that enables“gene swapping” reassortment. The haemagglutinin enables the virus tobind to host cell receptors and facilitates the entry of the virus intothe cell where it will replicate. The neuraminidase proteinenzymatically cleaves terminal sialic acid residues, and is believed toassist in the transport of the virus through the mucin layer of therespiratory tract as well as facilitating the budding of the progenyvirus away from the host cell. Influenza C viruses, which present muchless of a health-risk to humans possess a single surface protein whichcombines the haemagglutinin, fusion activity and receptor destroyingactivity.

As a result of the error prone RNA polymerase enzyme, both thehaemagglutinin and neuraminidase proteins of the influenza virus areliable to point mutations which need not necessarily affect the abilityof the virus to replicate. Such a mutation (or coincident mutations) atone of the sites recognised by the host antibody response may result inthe host antibody, induced by vaccination or a previous infection, beingunable to bind effectively to the “new” virus strain thereby allowing aninfection to persist. As the human influenza strains are continuallyevolving via these point mutations, the virus is able to escape from thelimited antibody repertoire of the human immune response and causeepidemics. The regular “seasonal” bouts of influenza infections aretherefore caused by the circulating strains in the population undergoingantigenic drift.

During seasonal epidemics influenza can spread around the world quicklyand inflicts a significant economic burden in terms of hospital andother healthcare costs and lost productivity. The virus is transmittedin droplets in the air from human-to-human and targets epithelial cellsin the trachea and bronchi of the upper respiratory tract. Influenzavirus may also be picked up from contaminated surfaces and passed to themouth. Disease spreads very quickly especially in crowded circumstancesthrough coughing and sneezing. The stability of the virus is favored bylow relative humidity and low temperatures and, as a consequence,seasonal epidemics in temperate areas tend to appear in winter. Greatermorbidity and mortality is observed with influenza A strains, withinfluenza B usually associated with lower attack rates and a milderdisease. Occasionally, however, influenza B can cause epidemics of thesame severity as type A viruses. Influenza B is primarily a childhoodpathogen and does not usually exhibit the same degree of antigenicvariation as type A.

The typical uncomplicated influenza infection is characterised by arapid onset of illness (headache, cough, chills) followed by fever, sorethroat, significant myalgias, malaise and loss of appetite. Furthersymptoms may include rhinorrhoea, substernal tightness and ocularsymptoms. The most prominent sign of infection is the fever that isusually in the 38-40° C. temperature range. Whilst the majority ofpeople will recover from influenza infection within one to two weekswithout requiring any medical treatment, for certain members of thepopulation the disease may present a serious risk. Such individualsinclude the very young, the elderly and people suffering from medicalconditions such as lung diseases, diabetes, cancer, kidney or heartproblems. In this “at risk” population, the infection may lead to severecomplications of underlying diseases, bacterial pneumonia, (caused byrespiratory pathogens such as Streptococcus pneumoniae, Haemophilusinfluenzae and Staphylococcus aureus) and death. The clinical featuresof influenza infection are similar in children, although their fever maybe higher and febrile convulsions can occur. In addition, children havea higher incidence of vomiting and abdominal pain as well as otitismedia complications, croup and myositis.

The World Health Organization estimates that in annual influenzaepidemics 5-15% of the population is affected with upper respiratorytract infections. Hospitalization and deaths mainly occur in high-riskgroups (elderly and the chronically ill). Although difficult to assess,these annual epidemics are thought to result in between three and fivemillion cases of severe illness and approximately 250 000 and 500 000deaths every year around the world. Over 90% of the deaths currentlyassociated with influenza in industrialized countries occur among theelderly over 65 years of age. In the U.S.A., the CDC estimate that morethan 200,000 people are hospitalized every year on average followingcomplications arising from seasonal influenza infection, with around36,000 excess mortalities being recorded.

The host immune response that controls the recovery from influenzainfection is conferred through a combination of serum antibodiesdirected to the surface proteins, mucosal secretory IgA antibodies andcell-mediated immune responses. About one to two weeks after a primaryinfection, neutralizing haemagglutination inhibiting (HAI) antibodies aswell as antibodies to neuraminidase are detectable in the serum, peakingat approximately three to four weeks. After re-infection, the antibodyresponse is more rapid. Influenza antibodies may persist for months oryears, although in some high-risk groups antibody levels can begin todecline within a few months after vaccination. Secretory IgA antibodiespeak approximately 14 days after infection and can be detected insaliva, nasal secretions, sputum and in tracheal washings. Preceding theoccurrence of antibody-producing cells, cytotoxic T lymphocytes withspecificity for influenza appear, and serve to limit the infection byreducing the maximal viral load whilst mediating more rapid viralclearance through the induction of antiviral cytokines and lysinginfected cells. In addition, mononuclear cells infiltrate infectedairways providing antibody dependent cell-mediated cytotoxicity againstinfluenza-infected cells.

To date, vaccine approaches against respiratory virus infections such asinfluenza essentially rely upon the induction of antibodies that protectagainst viral infection by neutralizing virions or blocking the virus'sentry into cells. These humoral immune responses target external viralsurface proteins that are conserved for a given strain.Antibody-mediated protection is therefore effective against homologousviral strains but inadequate against heterologous strains withserologically distinct surface proteins. This distinction is ofconsequence since the surface proteins of many viruses are capable ofrapid mutation; for example an effective humoral response-based vaccineagainst a form of the influenza virus may be ineffective against nextseason's variant.

There are currently two main types of licensed influenza vaccines. Onegroup of vaccines contains the haemagglutinin and neuraminidase surfaceproteins of the virus as the active immunogens. These include wholeinactivated virus vaccines, split virus vaccines consisting ofinactivated virus particles disrupted by detergent treatment, subunitvaccines consisting essentially purified surface proteins from whichother virus components have been removed and virosomes where the surfaceproteins are presented on a liposomal surface. The second groupcomprises the live attenuated, cold-adapted, strains of virus. For allthese vaccines a blend of surface antigens from usually three or fourvirus strains are required; current commercial influenza vaccinescontain antigens from two A subtypes, H3N2 and H1N1, and one type Bvirus. Each year in September and February respectively, the WHO GlobalInfluenza Program recommends the composition of the influenza vaccinefor the next season that normally begins in May-June in the southernhemisphere and in November-December in the northern hemisphere. Thecomposition is based on surveillance data from the worldwide network ofnational influenza centers and WHO collaborating centers and attempts tocover the likely strains to be circulating nine months later. For thisreason, manufacturers are obliged to change the composition of theinfluenza vaccine on an annual basis in order to ensure an accuratematch is achieved with the circulating viral strains.

Most inactivated influenza vaccines are given via the intramuscularroute in the deltoid muscle, except in infants where the recommendedsite is the antero-lateral aspect of the thigh. A single dose ofinactivated vaccine annually is appropriate, except for previouslyunvaccinated preschool children with pre-existing medical conditions whoshould receive two doses at least one month apart. The live attenuatedinfluenza vaccine (LAIV) is delivered intra-nasally. These have beenavailable in Russia for a number of years and recently licensed for usein the USA in pediatric populations. Such vaccines are able to elicitlocal antibody and cell-mediated immune responses at the nasalepithelial surface. The live attenuated influenza vaccine is not,however, licensed for use in the USA in elderly populations (over 50years old).

To enhance the breadth and intensity of the immune response mounted tothe influenza virus surface proteins, various adjuvants and alternativeimmuno-potentiating agents have been evaluated for inclusion in thevaccine formulation. An adjuvant in this context is an agent that isable to modulate the immune response directed to a co-administeredantigen while having few if any direct effects when given on its own.Recent licensed developments in the influenza vaccine field includeMF-59, a submicron oil-in water emulsion. Aluminium-containing adjuvantsare also used by some manufacturers. The intention of these adjuvants isto amplify the resulting serum antibody response to the administeredantigens.

Provided there is a good antigenic match between the vaccine strains andthose circulating in the general population, inactivated influenzavaccines prevent laboratory-confirmed illness in approximately 70%-90%of healthy adults. However, the CDC highlights that vaccine efficacy inthe elderly (over 65 years old) can be as low as 30-40%. Of relevance inthis regard is the observation that ageing in humans creates defects inmemory T-cell responses that reduce vaccine efficacy and increases therisk to natural infection. Furthermore, a clinical study in a communitybased setting demonstrated that cell mediated immunity, and not humoralimmunity, was correlated with influenza disease protection in a group ofover 60 year olds.

In addition, efficacy rates decline significantly if the vaccine strainis antigenically different to the circulating strains. Antigenicvariation studies have indicated that four or more amino acidsubstitutions over at least two antigenic sites of the influenza Ahaemagglutinin results in a drift variant sufficiently discrete toundermine a vaccine's efficacy (Jin et al. “Two residues in thehemagglutinin of A/Fujian/411/02-like influenza viruses are responsiblefor antigenic drift from A/Panama/2007/99.” Virology. 2005; 336:113-9).In a case controlled study of adults aged 50-64 years with laboratoryconfirmed influenza during the 2003-04 season when the vaccine andcirculating A/H3N2 strains were not well matched, vaccine effectivenesswas estimated to be 52% among healthy individuals and 38% among thosewith one or more high-risk conditions, according to the CDC. Thelikelihood of mismatching is raised by the limited manufacturing windowof opportunity; the time from strain confirmation, through seedproduction, antigen manufacture and purification, and the trivalentblending and product filling must all occur in typically less than sixmonths.

Occasionally, a new influenza strain emerges in the population with highpathogenicity and antigenic novelty which results in a worldwidepandemic. Pandemic influenza is the result of an antigenic shift in thesurface proteins and represents a serious threat to global health as nopre-existing immunity has been developed by individuals. Pandemicstrains are characterised by their sudden emergence in the populationand their antigenic novelty. During the twentieth century, fourpandemics occurred; in 1918 the causative strain was H1N1, in 1957 H2N2,in 1968 H3N2 and in 1977 H1N1.

There are three alternative explanations for the occurrence of antigenicshift. Firstly, as the influenza virus genome is segmented, it ispossible for two influenza strains to exchange their genes uponco-infection of a single host, for example swine, leading to theconstruction of a replication-competent progeny carrying geneticinformation of different parental viruses. This process, known asgenetic reassortment, is believed to have been the cause of the 1957 and1968 pandemics. The 1968 pandemic arose when the H3 haemagglutinin geneand one other internal gene from an avian donor reassorted with the N2neuraminidase and five other genes from the H2N2 human strain that hadbeen in circulation. Secondly, a non-human influenza strain acquires theability to infect humans. The 1918 pandemic arose when an avian H1N1strain mutated to enable its rapid and efficient transfer fromhuman-to-human. Thirdly, a strain that had previously caused an epidemicmay remain sequestered and unaltered within the human population. The1977 H1N1 pandemic strain, for example, was essentially identical to astrain that had caused an epidemic 27 years previously and wasundetected in the human and animal reservoir over the intervening years.

An influenza pandemic is threatened once three principal criteria havebeen met:

-   -   1. An influenza virus HA subtype, unseen in the human population        for at least one generation, emerges (or re-emerges).    -   2. The virus infects and replicates efficiently in humans,        causing significant illness.    -   3. The virus is transmitted readily and sustainably between        humans.

Global pandemics can afflict between 20% and 40% of the world'spopulation in a single year. The pandemic of 1918-19, for example,affected 200 million people, killing over 30 million worldwide. In theUnited States, more than half a million individuals died, whichrepresented 0.5% of the population. Although healthcare has dramaticallyimproved since that time, with vaccines and antiviral therapies beingdeveloped, the CDC estimate that a pandemic today would result in two toseven million deaths globally.

Since 1999, three different influenza subtype strains (H5N1, H7N7 andH9N2) have crossed from avian species to humans, all causing humanmortality. As of Aug. 14, 2007 a total of 320 human cases of H5N1 HighlyPathogenic Avian Influenza Virus (HPAIV) infection had been recordedworldwide, with 193 deaths.

Unlike normal seasonal influenza, where infection causes only mildrespiratory symptoms in most healthy people, the disease caused by H5N1follows an unusually aggressive clinical course, with rapiddeterioration and high fatality. Primary viral pneumonia and multi-organfailure are common. It is significant that most cases have occurred inpreviously healthy children and young adults. H5N1 HPAIV incubateslonger than other human influenza viruses before causing symptoms, up toeight days in some cases. In household clusters of cases, the timebetween cases has generally ranged from two to five days but has beenreported to take as long as 17 days.

Initial symptoms of H5N1 HPAIV infection are more likely to includediarrhea and can appear up to a week before any respiratory symptoms.This feature, combined with the detection of viral RNA in stool samples,suggests that the virus can multiply in the gastrointestinal tract.Lower respiratory tract symptoms such as shortness of breath appearearly in the course of the illness, whereas upper respiratory symptomssuch as rhinorrheoa are less common.

H5N1 HPAIV presently meets two of the conditions required for apandemic; the H5 haemagglutinin represents a new antigen for humans. Noone will have immunity should an H5N1-like pandemic virus emerge. Inaddition, the virus has infected more than 300 humans, with an apparentmortality rate of over 60%.

All prerequisites for the start of a pandemic have therefore been metsave one: the establishment of efficient and sustained human-to-humantransmission of the virus. The risk that the H5N1 virus will acquirethis ability will persist as long as opportunities for human infectionsoccur. This is believed to be a realistic probability, either throughstep-wise mutation or through reassortment with a human-adapted strain.

At the scientific level, one or more changes to the virus phenotype arenecessary before the virus strain could achieve ready human-to-humantransmission and begin a pandemic. However, a number of recentobservations including specific mutations detected in recent humanisolates from Turkey, the increasing pathogenicity to mammals of thecirculating virus, the expansion of the H5N1 HPAIV host range to includeother mammals, such as tigers and cats that were previously consideredto be resistant to infection with avian influenza viruses, all indicatethat the H5N1 virus is continuing to evolve capabilities that mayultimately facilitate human-to-human transmission.

Other influenza viruses with possibly even greater pandemic potentialmay yet emerge. These include a number of H9 and H7 virus strains, whichin recent years have also been transmitted to humans. H9 viruses are nowendemic in poultry in Asia and also have crossed efficiently into pigpopulations in South Eastern and Eastern China. Of concern is the factthat the H9N2 strains possess typical human-like receptor specificityand have a broad host range.

In early 2003, an H7N7 HPAIV outbreak occurred in poultry in theNetherlands. Bird-to-human transmission of the H7N7 virus occurred in atleast 82 cases. Conjunctivitis was the most common disease symptom inpeople infected with the H7 strain, with only seven cases displayingtypical influenza-like illness. The virus did not prove highlypathogenic for humans and only one fatal case was observed. Otherviruses with pandemic potential are those of the H2 subtype, because ofits past history as a pandemic virus, and H6 because of its highincidence in poultry species in Asia and North America.

This indicates that a threat of a new human influenza pandemic is notuniquely linked to the emergence of HPAI H5N1.

In preparation for an influenza pandemic a number of clinical trialswith candidate H5N1 influenza vaccines have been conducted. These haveconsistently shown that in order to generate a serum antibody responsepredicted to be protective, multiple doses of either a much higheramount of haemagglutinin antigen than is normally used in a seasonalvaccine or the inclusion of an adjuvant is required. This is a directreflection of the immunological naivety of the population to the H5haemagglutinin. At the present, the only options available for apandemic influenza vaccine are therefore either one with a very high HAcontent, which would severely limit the number of doses that could beproduced, or the use of an adjuvant that is not currently licensed inthe majority of countries. It should also be appreciated that a vaccinethat matches the pandemic strain will take many months to manufacturefrom the time that it is first isolated in humans; a stockpiled vaccineproduced in advance of the emergence of a pandemic will most probablynot be antigenically identical and therefore provide only limitedprotection, if any at all. Evidence of antigenic drift is alreadyevident in the most recent outbreaks of H5N1.

In summary, there is a clear requirement for both seasonal and pandemicinfluenza vaccines to be improved:

-   -   1. There are obvious limitations in their efficacy, in        particular in unprimed individuals. This is of specific concern        with regard to the prospects of an influenza pandemic arising        from antigenic shift.    -   2. The dependence on being able to predict accurately the        influenza strains likely to be circulating in the following        fall/winter seasons. A mismatch between the vaccine strains and        those actually causing infections will render a significant        proportion of the population vulnerable to influenza.    -   3. The need to re-vaccinate at risk groups on a yearly basis as        the virus undergoes antigenic drift.    -   4. Capacity constraints, as there are only a limited number of        potential biological manufacturing plants worldwide.    -   5. The protection afforded to the elderly age group is limited        by conventional vaccines.

Improved classes of influenza vaccine therefore are needed, which arepreferably synthetic, stable, and effective against all influenza Astrains (including potential pandemic strains), with enhanced efficacyin the elderly (at risk) groups.

SUMMARY OF THE INVENTION

The present invention seeks to overcome the problem of deliveringinfluenza antigens to immune responsive cells by using a fluorocarbonvector in order to enhance their immunogenicity. The fluorocarbon vectormay comprise one or more chains derived from perfluorocarbon or mixedfluorocarbon/hydrocarbon radicals, and may be saturated or unsaturated,each chain having from 3 to 30 carbon atoms.

Accordingly, in one aspect, the invention provides a fluorocarbonvector-antigen construct of structure C_(m)F_(n-)—C_(y)H_(x)-(Sp)-R orderivatives thereof, where m=3 to 30, n<=2m+1, y=0 to 15, x<=2y,(m+y)=3-30, Sp is an optional chemical spacer moiety and R is an antigenderived from the influenza virus. In another aspect, the inventionprovides a fluorocarbon vector-antigen construct of structure

where Sp is an optional chemical spacer moiety and R is an antigenderived from the influenza virus. These aspects of the invention canhave one or more of the following features. R can include one or moreepitopes from an influenza virus protein. R can include one or moreepitopes from an influenza virus type A protein or an influenza virustype B protein or an influenza virus type C protein. R can be a peptide,optionally an immunogenic peptide. R can be a peptide of between 7 to100 amino acids. R can include at least one MHC class I or II bindingepitope or B cell binding epitope, or combinations thereof. R caninclude two or more overlapping epitopes. R can be a peptide selectedfrom the NCBI and Los Alamos National Laboratory influenza sequencedatabases or fragments, derivatives, homologues or combinations thereof.R can be a peptide selected from SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, or 65 or fragments, derivatives, homologues or combinations thereof.The fluorocarbon vector can be non-covalently associated with anantigen. R can include multiple epitopes and/or fusion peptides.

Moreover, the present invention provides a pharmaceutical compositionthat includes one or more fluorocarbon vector-antigen constructsdescribed above, optionally together with one or more pharmaceuticallyacceptable carriers, excipients, diluents or adjuvants. Thepharmaceutical composition can be formulated for parenteral, oral,ocular, rectal, nasal, transdermal, topical, or vaginal administration.The pharmaceutical composition can take the form of a liquid, emulsion,solid, aerosol or gas. The pharmaceutical composition can be incombination with an adjuvant selected from: (1) natural or syntheticallyderived refinements of natural components of bacteria such as Freund'sadjuvant and its derivatives, muramyldipeptide (MDP) derivatives, CpG,monophosphoryl lipid A; (2) adjuvants or potentiating agents such assaponins, aluminium salts and cytokines; (3) oil in water adjuvants,water-in-oil adjuvants, immunostimulating complex (ISCOMs), liposomes,formulated nano and micro-particles; and (4) bacterial toxins andtoxoids. The pharmaceutical composition can include more than one (e.g.at least two) vector-antigen constructs, and the first construct caninclude the influenza peptide sequence:

(SEQ ID NO: 1) HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKand the second construct comprising the influenza peptide sequence:

(SEQ ID NO: 17) YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE.For example, the pharmaceutical composition can include 8 vector-antigenconstructs that include the following influenza peptide sequences:

Construct 1 - (SEQ ID NO: 1) HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADK;Construct 2 - (SEQ ID NO: 4) VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQG;Construct 3 - (SEQ ID NO: 17) YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE;Construct 4 - (SEQ ID NO: 18) APIMFSNKMARLGKGYMFESKXMKLRTQIPAEMLA, whereX can be R or S; Construct 5 - (SEQ ID NO: 19)SPGMMMGMFNMLSTVLGVSILNLGQKKYTKTTY; Construct 6 - (SEQ ID NO: 20)KKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFG; Construct 7 - (SEQ ID NO: 32)DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS; Construct 8 - (SEQ ID NO: 35)DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER.

The pharmaceutical composition can be administered in combination with ahumoral response-based influenza vaccine, either contemporaneously orseparately. For example, the pharmaceutical composition can be ahaemagglutinin containing influenza vaccine.

In another aspect, the invention provides a use of one or more of thefluorocarbon vector-antigen constructs described above in thepreparation of a prophylactic vaccine or immunotherapeuticpharmaceutical product. The prophylactic vaccine or immunotherapeuticpharmaceutical product can be for parenteral, mucosal, oral, nasal,topical, ocular, rectal, transdermal, or vaginal administration. Themethod of preparing a prophylactic or therapeutic pharmaceutical productcan include combining one or more fluorocarbon constructs describedabove with one or more pharmaceutically acceptable carriers, excipients,diluents, or adjuvants, optionally for parenteral, mucosal, oral, nasal,topical, ocular, rectal, transdermal, or vaginal administration.

In other aspect, the invention provides a method of using one or more ofthe compositions described above. This aspect of the invention can haveone or more of the following features. The method can include treatmentor immunization using any of the compositions described above. Themethod can include stimulating an immune response, includingadministering the formulation of any of the compositions described aboveto an animal, such as a bird, a mammal, or a human. The method caninclude administering a pharmaceutical composition described above, incombination with anti-influenza therapy, such as administration of aneuraminidase inhibitor. The method can include stimulating an immuneresponse by administering any of the preventative or therapeuticformulations described above, in combination with a haemagglutinincontaining influenza vaccine, either contemporaneously or separately.

It should be understood that different features and embodiments of theinvention, including those described under different aspects of theinvention, are meant to be generally applicable to all aspects of theinvention. Any embodiment may be combined with any other embodimentunless inappropriate. All examples are illustrative and non-limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and embodiments of the invention may be more fullyunderstood by reference to the following detailed description andclaims.

FIG. 1 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus its native peptide equivalent in BALB/c andCBF6 mice, after prime or prime-boost, assessed by ex vivo IFN-γ ELIspotassay.

FIG. 2 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus its native peptide equivalent in BALB/c andCBF6 mice, after prime or prime-boost, assessed by ex vivo IFN-γ ELIspotassay.

FIG. 3 shows a comparison of individual peptide immunogenicity offluoropeptides verses native peptides in BALB/c and CBF6 mice afterprime or prime-boost assessed by ex vivo IFN-γ ELISpot.

FIG. 4 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus the native peptide equivalent in BALB/c andCBF6 mice after prime-boost immunization, assessed by cytokine profiles.

FIG. 5 is a graph showing that both CD4+ T cells and CD8+ T cells arestimulated by the fluoropeptide vaccine in BALB/c mice.

FIG. 6 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus vaccine emulsified in CFA in BALB/c miceafter a single immunization; assessment of cytokine profiles.

FIG. 7 shows a comparison of subcutaneous versus intradermal routes offluoropeptide vaccine administration in BALB/c mice after a singleimmunization: ex vivo IFN-γ ELISpot assay.

DETAILED DESCRIPTION

Over the decades numerous delivery methods have been evaluated,including vectors such as Penetratin, TAT and its derivatives, DNA,viral vectors, virosomes and liposomes. However, these systems eitherelicit very weak CTL responses, fail to generate a booster amplificationon memory responses, have associated toxicity issues or are complicatedand expensive to manufacture at the commercial scale. There is thereforea recognised need for improved vectors to direct the intracellulardelivery of antigens in the development of vaccines and drugs intendedto elicit a cellular immune response. A vector in the context ofimmunotherapeutics or vaccines is any agent capable of transporting ordirecting an antigen to immune responsive cells in a host.

The present invention seeks to overcome the problem of deliveringinfluenza antigens to immune responsive cells by using a fluorocarbonvector in order to enhance their immunogenicity. The fluorocarbon vectormay comprise one or more chains derived from perfluorocarbon or mixedfluorocarbon/hydrocarbon radicals, and may be saturated or unsaturated,each chain having from 3 to 30 carbon atoms.

Fluorinated surfactants have low critical micelle concentrations andthus self-organise into multimolecular micelle structures at a lowconcentrations. This physicochemical property is related to the stronghydrophobic interactions and low Van der Waal's interactions associatedwith fluorinated chains which dramatically increase the tendency offluorinated amphiphiles to self-assemble in water and to collect atinterfaces. The formation of such structures facilitates their endocyticuptake by cells, to for example antigen-presenting cells (Reichel F. etal. J. Am. Chem. Soc. 1999, 121, 7989-7997). Furthermore haemolyticactivity is strongly reduced and often suppressed when fluorinatedchains are introduced into a surfactant (Riess et al. Adv. Mater. 1991,3, 249-251) thereby leading to a reduction in cellular toxicity.

In order to link the vector to the antigen through a covalent linkage, areactive group, or ligand, is incorporated as a component of the vector,for example —CO—, —NH—, S, O or any other suitable group is included;the use of such ligands for achieving covalent linkages are well-knownin the art. The reactive group may be located at any position on thefluorocarbon molecule.

Coupling of the fluorocarbon vector to the antigen may be achievedthrough functional groups such as —OH, —SH, —COOH, —NH₂ naturallypresent or introduced onto any site of the antigen. Suitable links maycontain a nitrogen, oxygen or sulphur atom, in either linear or cyclicform. Examples of the bonds formed by ligation may include oxime,hydrazone, disulphide or triazole or any suitable covalent bond. Inparticular, the fluorocarbon moiety could be introduced through athioester bond to increase the immunogenicity of the peptide (Beekman etal. “Synthetic peptide vaccines: palmitoylation of peptide antigens by athioester bond increases immunogenicity.” J. Peptide Res. 1997, 50,357-364). Optionally, a spacer element (peptidic, pseudopeptidic ornon-peptidic) may be incorporated to permit cleavage of the antigen fromthe fluorocarbon element for processing within the antigen-presentingcell and to optimise antigen presentation, as previously shown forlipopeptides (Verheul et al. “Monopalmitic acid-peptide conjugatesinduce cytotoxic T cell responses against malarial epitopes: importanceof spacer amino acids,” Journal of Immunological Methods 1995, volume182, pp 219-226).

In a first aspect, the present invention provides a fluorocarbonvector-antigen construct having a chemical structureC_(m)F_(n-)C_(y)H_(x)-(Sp)-R or derivatives thereof, where m=3 to 30,n<=2m+1, y=0 to 15, x<=2y, (m+y)=3-30 and Sp is an optional chemicalspacer moiety and R is an antigen derived from the influenza virus.

In the context of the present invention “derivatives” refers torelatively minor modifications of the fluorocarbon compound such thatthe compound is still capable of delivering the antigen as describedherein. Thus, for example, a number of the fluorine moieties can bereplaced with other halogen moieties such as chlorine (CO, if) bromine(Br) or iodine (I). In addition it is possible to replace a number ofthe fluorine moieties with methyl groups and still retain the propertiesof the molecule as discussed herein.

In a particular example of the above formula the vector may be 2H, 2H,3H, 3H-perfluoroundecanoic acid of the following formula:

Thus in a second aspect the invention provides a fluorocarbonvector-antigen construct of structure

where Sp is an optional chemical spacer moiety and R is an antigenderived from the influenza virus.

As used herein the term “antigen” refers to a molecule having theability to be recognized by immunological receptors such as T cellreceptor (TCR) or B cell receptor (BCR or antibody). Antigens may beproteins, protein subunits, peptides, carbohydrates, lipid orcombinations thereof, natural or non-natural, provided they present atleast one epitope, for example a T cell and/or a B cell epitope.

Such antigens may be derived by purification from the native protein orproduced by recombinant technology or by chemical synthesis. Methods forthe preparation of antigens are well-known in the art. Furthermore,antigens also include DNA or oligonucleotide encoding an antigenicpeptide or protein.

The antigen associated with the vector may be any influenza antigencapable of inducing an immune response in an animal, including humans.Preferably the immune response will have a beneficial effect in thehost.

The influenza antigen may contain one or more T cell epitopes or one ormore B cell epitopes or combinations of T and B cell epitopes.

The T cell epitopes may be MHC class I or class II restricted.

As used herein the term “epitope” includes:

-   -   (i) CD4+ T cell epitopes which are peptidic sequences containing        an MHC class II binding motif and having the ability to be        presented at the surface of antigen presenting cells by MHC        class II molecules, and    -   (ii) CD8+ T cell epitopes which are peptidic sequences        containing an MHC class I binding motifs and having the ability        to be presented by MHC class I molecules at the cell surface,        and    -   (iii) B cell epitopes which are peptidic sequences having a        binding affinity for a B cell receptor.

The antigen may comprise one or more epitopes from an influenza type Aprotein, an influenza type B protein or an influenza type C protein.Examples of the influenza virus proteins, from both the influenza A andB types, include: haemagglutinin, neuraminidase, matrix (M1) protein,M2, nucleoprotein (NP), PA, PB1, PB2, NS1 or NS2 in any suchcombination.

Thus in a further aspect, the present invention provides avector-antigen construct where the influenza virus antigen is a protein,protein subunit, peptide, carbohydrate or lipid or combinations thereof.For the construct to be immunologically active the antigen must compriseone or more epitopes. Preferably the antigen is a peptide sequencederived from the influenza virus. Peptides or proteins of the inventionpreferably contain a sequence of at least seven, more preferably between9 and 100 amino-acids and most preferably between around 15 to 40 aminoacids. Preferably, the amino acid sequence of the epitope(s) bearingpeptide is selected to enhance the solubility of the molecule in aqueoussolvents. Furthermore, the terminus of the peptide which does notconjugate to the vector may be altered to promote solubility of theconstruct via the formation of multi-molecular structures such asmicelles, lamellae, tubules or liposomes. For example, a positivelycharged amino acid could be added to the peptide in order to promote thespontaneous assembly of micelles. Either the N-terminus or theC-terminus of the peptide can be coupled to the vector to create theconstruct. To facilitate large scale synthesis of the construct, the N-or C-terminal amino acid residues of the peptide can be modified. Whenthe desired peptide is particularly sensitive to cleavage by peptidases,the normal peptide bond can be replaced by a non-cleavable peptidemimetic; such bonds and methods of synthesis are well known in the art.

Non-standard, non-natural amino-acids can also be incorporated inpeptide sequences provided that they do not interfere with the abilityof the peptide to interact with MHC molecules and remain cross-reactivewith T cells recognizing the natural sequences. Non-natural amino-acidscan be used to improve peptide resistance to protease or chemicalstability. Examples of non-natural amino acids include the D-amino-acidsand cysteine modifications.

More than one antigen may be linked together prior to attachment to thefluorocarbon vector. One such example is the use of fusion peptideswhere a promiscuous T helper epitope can be covalently linked to one ormultiple CTL epitopes or one or multiple B cell epitope which can be apeptide, a carbohydrate, or a nucleic acid. As an example, thepromiscuous T helper epitope could be the PADRE peptide, tetanus toxoidpeptide (830-843) or influenza haemagglutinin, HA (307-319).Alternatively, the peptide sequence may contain two or more epitopes,which may be overlapping thereby creating a cluster of densely packedmulti-specific epitopes, or contiguous, or separated by a stretch ofamino acids.

Thus in a further aspect, the present invention provides avector-antigen construct where R is more than one epitope or antigenlinked together. Epitopes may also be linear overlapping therebycreating a cluster of densely packed multi-specific epitopes.

Due to the strong non-covalent molecular interactions characteristic tofluorocarbons, the antigen may also be non-covalently associated withthe vector and still achieve the aim of being favorably taken up byantigen-presenting cells.

Thus in a further aspect, the present invention provides avector/antigen construct where the antigen is non-covalently associatedwith the fluorocarbon vector.

Antigens bearing one or more B-cell epitopes may also be attached to thefluorocarbon vector, either with or without one or more T-cell epitopes.B cell epitopes can be predicted using in silico approaches (Bublil etal. “Stepwise prediction of conformational discontinuous B-cell epitopesusing the Mapitope algorithm.” Proteins. 2007 Jul. 1; 68(1):294-304.Greenbaum et al. “Towards a consensus on datasets and evaluation metricsfor developing B-cell epitope prediction tools” J Mol Recognit. 2007March-April; 20(2):75-82).

The present invention also provides vaccines and immunotherapeuticscomprising one or more fluorocarbon vector-antigen constructs.Multi-component products of this type are desirable since they arelikely to be more effective in eliciting appropriate immune responses ina greater number of individuals. Due to extreme HLA polymorphism inhumans, it is unlikely that a single fluoropeptide will induce amultiepitopic immune response in a high percentage of a givenpopulation. Therefore, in order for a vaccine product to be effectiveacross a population a number of fluoropeptides may be necessary in thevaccine formulation in order to provide broad coverage. Moreover, theoptimal formulation of an influenza vaccine or immunotherapeutic maycomprise a number of different peptide sequences derived from differentinfluenza virus antigens. In this case the peptides may be linkedtogether attached to a single fluorocarbon vector or each peptideantigen could be bound to a dedicated vector.

A multi-component product may contain one or more vector-antigenconstructs, more preferably 2 to about 20, more preferably 3 to about10. In particular embodiments the multi component vaccine may contain 5,6, 7 or 8 eight constructs. This ensures that a multi-epitopic T-cellresponse is generated with a broad population coverage (i.e., addressesHLA diversity). For example, a formulation of multiple fluoropeptidesmay be composed of influenza A derived peptides alone, influenza Bderived peptides alone or influenza C derived peptides alone orcombinations of influenza types, most preferably influenza A and B.

In one embodiment the product comprises at least two vector-antigenconstructs, the first construct comprising the influenza peptidesequence:

(SEQ ID NO: 1) HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKand the second construct comprising the influenza peptide sequence:

(SEQ ID NO: 17) YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE

In a further embodiment the product comprises 8 vector-antigenconstructs which comprise the following influenza peptide sequences:

Construct 1 - (SEQ ID NO: 1) HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKConstruct 2 - (SEQ ID NO: 4) VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGConstruct 3 - (SEQ ID NO: 17) YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFEConstruct 4 - (SEQ ID NO: 18) APIMFSNKMARLGKGYMFESKXMKLRTQIPAEMLA, whereX can be R or S Construct 5 - (SEQ ID NO: 19)SPGMMMGMFNMLSTVLGVSILNLGQKKYTKTTY Construct 6 - (SEQ ID NO: 20)KKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFG Construct 7 - (SEQ ID NO: 32)DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS Construct 8 - (SEQ ID NO: 35)DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER

Alternatively, multiple epitopes may be incorporated into a formulationin order to confer immunity against a range of pathogens, one of whichis the influenza virus. For example a respiratory infection vaccine maycontain antigens from influenza virus and respiratory syncytial virus.

Compositions of the invention comprise fluorocarbon vectors associatedto antigens optionally together with one or more pharmaceuticallyacceptable carriers and/or adjuvants. Such adjuvants and/orpharmaceutically acceptable carriers, would be capable of furtherpotentiating the immune response both in terms of magnitude and/orcytokine profile, and may include, but are not limited to:

-   -   (1) natural or synthetically derived refinements of natural        components of bacteria such as Freund's adjuvant & its        derivatives, muramyldipeptide (MDP) derivatives, CpG,        monophosphoryl lipid A;    -   (2) other known adjuvant or potentiating agents such as        saponins, aluminium salts and cytokines;    -   (3) methods of formulating antigens with or without extraneous        adjuvants (see 1 & 2 above) such as oil in water adjuvants,        water-in-oil adjuvants, immunostimulating complex (ISCOMs),        liposomes, formulated nano and micro-particles;    -   (4) bacterial toxins and toxoids; and    -   (5) other useful adjuvants well-known to one skilled in the art.

The choice of carrier if required is frequently a function of the routeof delivery of the composition. Within this invention, compositions maybe formulated for any suitable route and means of administration.Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, ocular, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, transdermal)administration.

The formulation may be administered in any suitable form, for example asa liquid, solid, aerosol, or gas. For example, oral formulations maytake the form of emulsions, syrups or solutions or tablets or capsules,which may be enterically coated to protect the active component fromdegradation in the stomach. Nasal formulations may be sprays orsolutions. Transdermal formulations may be adapted for their particulardelivery system and may comprise patches. Formulations for injection maybe solutions or suspensions in distilled water or anotherpharmaceutically acceptable solvent or suspending agent.

Thus in a further aspect, the present invention provides a prophylacticor therapeutic formulation comprising the vector-antigen construct(s)with or without a suitable carrier and/or adjuvant.

The appropriate dosage of the vaccine or immunotherapeutic to beadministered to a patient will be determined in the clinic. However, asa guide, a suitable human dose, which may be dependent upon thepreferred route of administration, may be from 1 to 1000 μg. Multipledoses may be required to achieve an immunological or clinical effect,which, if required, will be typically administered between 2 to 12 weeksapart. Where boosting of the immune response over longer periods isrequired, repeat doses 1 month to 5 years apart may be applied.

The formulation may combine the vector-antigen construct with anotheractive component to effect the administration of more than one vaccineor drug. A synergistic effect may also be observed through theco-administration of the two or more actives.

A vaccine formulation of the invention, comprising one or morefluoropeptides, may be used in combination with a humoral response-basedinfluenza vaccine, such as Fluzone®, Agrippal™, Begrivac™, Fluvax®,Enzira®, Fluarix™, Flulaval™, FluAd®, Influvac®, Fluvirin®, FluBlok® orany influenza vaccine comprising haemagglutinin as the active component,or a live attenuated influenza virus, including the cold-adapted strainssuch as Flumist®. Administration may be as a combined mixture or asseparate vaccine agents administered contemporaneously or separated bytime.

In a further aspect the influenza vaccine formulation may beadministered in combination with an anti-viral therapeutic composition,including neuraminidase inhibitor treatments such as amanidine,rimantidine, zanamivir or oseltamivir. Administration may becontemporaneous or separated by time.

In other aspects the invention provides:

i) Use of the immunogenic construct as described herein in thepreparation of a medicament for treatment or prevention of a disease orsymptoms thereof.ii) A method of treatment through the induction of an immune responsefollowing administration of the formulation described herein.

Role of T Cells in Protection Against Influenza Disease

Whilst conventional influenza vaccine technologies have focusedprimarily on the antibody responses to the viral surface proteins, theseare subject to antigenic shift and drift which undermines efficacy andcreates the logistical vulnerabilities described. In contrast, T cells,which mediate cellular immune responses, can target proteins more highlyconserved across heterologous viral strains and clades. This propertygives vaccines that induce protective cellular immune responses thepotential to protect against heterologous viral strains and clades(heterosubtypic immunity). For the influenza virus, conservation of thePB1, PB2, PA, NP, M1, M2, NS1 and NS2 proteins and persistence of thecorresponding antigen-specific CD4+ and CD8+ T cells makes theseproteins attractive vaccine targets.

Protective antiviral cell-mediated immunity consists of the induction ofa Type 1 response supported by Type 1 CD4+ T-helper lymphocytes (Th1)leading to the activation of immune effector mechanisms including theinduction and maintenance of cytotoxic T lymphocytes (CTLs) as well asimmunostimulatory cytokines such as IFN-γ and IL-2. The CD4+ T helpercells are primarily responsible for helping other immune cells throughdirect cell-cell interactions or by secreting cytokines afterrecognizing antigenic T cell peptide epitopes bound to majorhistocompatibility complex (MHC) class II molecules. The cytotoxic Tlymphocytes (CTLs) typically express CD8 and induce lysis or apoptosisof cells on which they recognize foreign antigens presented by MHC classI molecules, providing a defense against intracellular pathogens such asviruses. This association of phenotype and function is not absolute,since CD4+ cells may exhibit cytolytic activity, while CD8+ cellssecrete antiviral cytokines, notably interferon-γ (IFN-γ) and tumornecrosis factor. Indeed, CD4⁺ CTL activity has been proposed as anotherimmune mechanism to control acute and chronic viral infection in humans.CD4⁺ CTL may control viral spread by direct antiviral cytolytic effectand may play a direct antiviral activity by the production of antiviralcytokines such as IFN-γ. IFN-γ is known to have a direct inhibitory andnon-cytolytic effect on virus production. CD4+ T helper cells are alsoessential in determining B cell antibody response and class switching,and in maximizing bactericidal activity of phagocytes such asmacrophages.

Cellular immune responses are believed to play an important role incontrolling influenza infection, ameliorating signs of disease andpromoting disease recovery. Influenza-specific cellular immunity iselicited following natural infection and several viral proteins havebeen identified as targets for human memory heterosubtypic T cellresponses, including nucleoprotein (NP), polymerase (PB1, PB2, & PA), M1and M2 proteins, and non-structural protein-1 (NS1). NS2 may also beimplicated. These internal proteins contain highly conserved andimmunodominant regions making them ideal T cell targets. In particular,experimental studies have shown that influenza-A NP represents animportant target antigen for both subtype-specific and cross-reactiveCTLs in mice and humans. This contrasts with haemagglutinin (HA) andneuraminidase (NA), which are unsuitable targets due to their highsequence variability within and between influenza subtypes.

More specifically, cell-mediated immunity is strongly implicated in theprotection against influenza disease including highly pathogenicstrains. Memory CD4+ and CD8+ T cells are present in the lung airwaysand evidence is mounting that these cells play a role in pulmonaryimmunity to influenza challenge by mediating engagement of the pathogenat the site of infection when pathogen loads are low. Depletion of CD8+T cells reduces the capacity of primed mice to respond to influenzainfection, which signifies a role for CD8+ T cells in the protectivesecondary response. Because viral replication is confined to cells inthe respiratory epithelium, CD8+ T cells exert their effector functionsat this site, producing antiviral cytokines and lysing target cellspresenting viral determinants for which they bear a specific T-cellreceptor. Lysis of infected epithelial cells is mediated by exocytosisgranules containing perforin and granzyme, as well as Fas mechanisms.(Thomas et al. “Cell-mediated protection in influenza infection.” EmergInfect Dis. 2006 January; 12(1):48-54).

Vigorous CD4+ T cell responses to influenza are initiated in thedraining lymph node followed by the spleen and they peak in the lung andbronchoalveolar secretions at day 6-7 post infection. This primary CD4T-cell response to influenza infection, albeit smaller in magnitude thanthe CD8 response, has been shown to involve robust CD4+ expansion, Th-1differentiation and their migration to the site of infection. CD4+T-helper cells are also necessary for long lasting and effective CD8memory to influenza infection. CD4 effector T-cell and memory responsescontribute to immunity against influenza via multiple mechanismsincluding their classic contribution as helpers during the generation ofinfluenza specific CD8+ CTL responses, their ability to drive IgG2a toneutralize infective viral particles, and via their direct antiviralactivity through the secretion of IFN-gamma. Both CD4+ and CD8+ T-cellepitopes have been shown to promote viral clearance and conferprotection in mice against an influenza challenge.

Mouse models for influenza-A virus provide an experimental system toanalyze T-cell mediated immunity. In particular, the T-cell immuneresponse to influenza infection has been well characterized in C57BL/6(H2^(b)) and Balb/C (H2^(d)) mice and their hybrids. Plotnicky et al.“The immunodominant influenza matrix T cell epitope recognized in humaninduces influenza protection in HLA-A2/K(b) transgenic mice.” Virology.2003 May 10; 309(2):320-9.) demonstrated the protective efficacy of theinfluenza matrix protein (M1) epitope 58-66 to lethal transgenic murinechallenge. Protection was mediated by T-cells since protection wasabolished following in vivo depletion of CD8+ and/or CD4+ T-cells. Mousesurvival correlated with M1-specific T-cells in the lungs, which weredirectly cytotoxic to influenza-infected cells following influenzachallenge. Woodland et al. “Identification of protective andnon-protective T cell epitopes in influenza.” Vaccine. 2006 Jan. 23;24(4):452-6) also demonstrated that a single CD4+ T cell epitope HA(211-225) could confer partial control of viral infection in vaccinatedmice.

Whilst T cell targets tend to be prone to less frequent mutation thanthe influenza virus surface protein B cell epitopes, CD8+ and CD4+ Tcell epitopes will also mutate under protective immune pressure overtime (Berkhoff et al. “Fitness costs limit escape from cytotoxic Tlymphocytes by influenza A viruses.” Vaccine. 2006 Nov. 10;24(44-46):6594-6.). This escape likely results from the confrontationbetween the virus and the highly polymorphic human leukocyte antigen(HLA) class I and II proteins which determines antigen processing andepitope presentation to host CD8⁺ and CD4+ T-cells respectively. Thisviral escape mechanism has been more clearly established for HIV and HCVand is known to shape the evolution of the virus. Therefore theselection of highly conserved peptide sequences with low inherentvariability (entropy) is an important factor to be considered in thedesign of T-cell vaccines which can specifically counter antigenic shiftand drift. Such methods have been described by Berkhoff et al.“Functional constraints of influenza A virus epitopes limit escape fromcytotoxic T lymphocytes” J Virol. 2005 September; 79(17):11239-46.)

Adults over 65 years of age currently account for approximately 90% ofall influenza-related mortality. This is also the target group wherecurrent vaccines are least effective. In humans, ageing appears to beassociated with a decline in the ability to generate T-cell effectorsfrom memory sub-populations. An increased frequency of central memoryCD4+ T-cells and decreased frequency of effector memory CD4+ T cells inthe elderly post-vaccination has been observed, which may be related todecreased levels of serum IL-7. Elderly subjects also demonstrate ablunted type-1 T-cell response to influenza vaccination which correlatesdirectly with IgG1 responses. Furthermore, mice also exhibit an agerelated impairment of epitope-specific CD8+ CTL activity during primaryinfluenza-A infection. This is associated with a defect in expansion ofCD8+ CTL rather than effector activity of influenza-specific CD8+ Tcells. (Mbawuike et al. “Reversal of age-related deficient influenzavirus-specific CTL responses and IFN-gamma production by monophosphoryllipid A.” Cell Immunol. 1996 Oct. 10; 173(1):64-78.)

As an important element of the T cell response is directed at theclearance of infected cells, a T-cell vaccine may be used in aprophylactic manner to generate memory recall as well as in atherapeutic mode, post-infection, to enhance the host's naturalcell-mediated immunity. The T-cell vaccine may also be used incombination with a conventional antibody-generating (humoralresponse-based) influenza vaccine, either through co-administration orby separate administration.

T-Cell Vaccine Approaches

A review of the T-cell and Influenza vaccine fields highlights a numberof critical challenges faced in the design of a broadly cross protectiveT-cell vaccine. A T-cell vaccine must first be capable of priming andboosting CD4+ HTL and CD8+ CTL T-cell memory and effector functions in ahigh percentage of vaccine recipients. Such a vaccine must also addressviral genetic diversity, and ongoing mutation, as well as human geneticdiversity manifest at the level of MHC allele polymorphism. The proposedinvention seeks to address these design issues by combining a novelfluoropeptide vaccine delivery system together with highly conservedinfluenza peptides. The peptides are preferably antigens known tocontain one or more epitopes, in particular T-cell epitopes.

Traditional peptide-based T-cell vaccine approaches have beenepitope-based and focussed on minimal CTL (8-11aa) or T-helper (13aa)epitopes delivered as single epitopes or reconstituted artificialstrings. Non-natural sequences may face inefficient antigen processingconstraints as well as giving rise to the potential formation ofunrelated neo-epitopes. Long, natural conserved peptide sequencescontaining overlapping T-cell epitopes, clustered T-cell epitopes orpromiscuous T-cell epitopes in a single peptide sequence permit naturalantigen processing while achieving broad population coverage. Moreover,the use of multiples of these long natural peptides in the one vaccineformulation is likely to offer even greater population coverage.Precedent shows long peptides (30-35aa) comprising CD4+& CD8+ T-cellepitopes have the ability to induce multi-epitopic responses in animalsand humans (Coutsinos et al. “Long-term specific immune responsesinduced in humans by a human immunodeficiency virus type 1 lipopeptidevaccine: characterization of CD8+-T-cell epitopes recognized.” J Virol.2003 October; 77(20):11220-31.). For an effective anti-viral CTLresponse (CD8 T cell driven), an appropriate Th-1 cytokine environmentis required (ensured by CD4 cells), thus the concomitant delivery of CD4and CD8 epitopes is predicted to enhance cellular responses (Krowka etal. “A requirement for physical linkage between determinants recognizedby helper molecules and cytotoxic T cell precursors in the induction ofcytotoxic T cell responses” J. Immunol 1986, May 15; 136(10):3561-6.).

CD4+ and CD8+ T cells recognize short peptides resulting from theextracellular and intracellular processing of foreign and self proteins,presented bound to specific cell surface molecules encoded by the MHCsystem. There are two discrete classes of MHC molecules: (i) MHC class Ipresents endogenous peptides; and (ii) MHC class II presents exogenouspeptides. The process of MHC class I antigen presentation involvesprotein degradation, peptide transport to the endoplasmic reticulum,peptide-MHC binding and export of peptide-MHC complexes to the cellsurface for recognition by CD8+ T cells. Peptides are bound within aspecific MHC binding groove, the shape and characteristics of whichresults in the binding of specific subsets of peptides sharing a commonbinding motif T cells are activated when the T-cell receptor recognizesa specific peptide-MHC complex, and in this way identify cells infectedby intracellular parasites or viruses or cells containing abnormalproteins (e.g. tumor cells) and mount appropriate immune responsesagainst them.

The peptides involved in specific peptide-MHC complexes triggeringT-cell recognition (T-cell epitopes) are important tools for thediagnosis and treatment of infectious, autoimmune, allergic andneoplastic diseases. Because T-cell epitopes are subsets of MHC-bindingpeptides, precise identification of portions of proteins that can bindMHC molecules is important for the design of vaccines andimmunotherapeutics. The MHC polymorphism is very high in the humanpopulation with 580 HLA-A, 921 HLA-B, 312 HLA-C, 527 HLA-DR(beta), 127HLA-DRQ(beta) and 86 HLA-DQ(beta) alleles known to date. This situationis challenging when having to design a T-cell based vaccine with broadpopulation coverage. MHC-binding peptides contain position-specificamino acids that interact with the groove of the MHC molecule(s),contributing to peptide binding. The preferred amino acids at eachposition of the binding motif may vary between allelic variants of MHCmolecules. Computational models facilitate identification of peptidesthat bind various MHC molecules. A variety of computational methods, MHCbinding assays, X-ray crystallography study and numerous other methodsknown in the art permit the identification of peptides that bind to MHCmolecules. Novel in silico antigen identification methodologies offerthe ability to rapidly process the large amounts of data involved inscreening peptide sequences for HLA binding motifs necessary todelineate viral sequences useful for a T cell vaccine. HLA basedbioinformatics approaches have been successfully applied in many fieldsof immunology and make it possible to address human genetic diversityconcerns, for example: Depil et al. “Determination of a HLA IIpromiscuous peptide cocktail as potential vaccine against EBV latency IImalignancies.”, J Immunother (1997). 2007 February-March; 30(2):215-26;Frahm et al. “Extensive HLA class I allele promiscuity among viral CTLepitopes.” Eur J Immunol. 2007 Aug. 17; 37(9):2419-2433; Schulze zurWiesch et al. “Broad repertoire of the CD4+ Th cell response inspontaneously controlled Hepatitis C virus infection includes dominantand highly promiscuous epitopes.” J Immunol. 2005 Sep. 15;175(6):3603-13; Doolan et al. “HLA-DR-promiscuous T cell epitopes fromPlasmodium falciparum pre-erythrocytic-stage antigens restricted bymultiple HLA class II alleles.” J Immunol. 2000 Jul. 15;165(2):1123-37.).

Peptides that bind more than one MHC allelic variant (‘promiscuouspeptides’) are prime targets for vaccine and immunotherapy developmentbecause they are relevant to a greater proportion of the humanpopulation. Promiscuous CD4+ T cell epitopes were also reported to bindmultiple MHC class II molecules. (Panina-Bordignon et al. “Universallyimmunogenic T cell epitopes: promiscuous binding to human MHC class IIand promiscuous recognition by T cells.” Eur J Immunol. 1989 December;19(12):2237-42.) On the other hand, some promiscuous CD8+ T cellepitopes were previously described having the ability to bind multiplesMHC class I molecules sharing binding characteristics and forming aso-called supertype (Frahm et al. “Extensive HLA class I allelepromiscuity among viral CTL epitopes.” Eur J Immunol. 2007 Aug. 17;37(9):2419-2433; Sette et al. ‘HLA supertypes and supermotifs: afunctional perspective on HLA polymorphism.’ Curr Opin Immunol. 1998August; 10(4):478-82). The identification of promiscuous CD4+ and CD8+ Tcell epitopes represent an important strategy in vaccine design in orderto achieve broad population coverage. MHC polymorphism is also addressedby selecting peptides known or predicted to contain an MHC binding motifrelated to highly frequent MHC alleles in a specific ethnic group oracross multiple ethnic groups.

By selecting a combination of sequences that provide broad populationcoverage and are conserved across a range of influenza strains(identified by using, for example, the National Center for BiotechnologyInformation (NCBI) or Los Alamos National Laboratory (LANL) influenzasequence databases) one is able to address viral genetic diversity andachieve protection against the majority, if not all, relevant influenzastrains.

Historically, the key failings of T-cell vaccine technologies (DNA andviral vector vaccines) have been the low percentage of vaccine subjectsresponding to the vaccines, often low levels of immunogenicity and theirability to achieve a booster amplification of memory and effector T-cellresponses. The principal goal for an effective influenza T-cell vaccineis to promote robust T-cell memory responses such that on re-exposure toantigen there is rapid expansion of effector functions which controlviral load and promote viral clearance from the lungs. To achieve this,robust virus specific Th-1 directed CD4+& CD8+ T-cell central andeffector memory responses are required. For a viable, commercial productthis response must be elicited in a high percentage of vaccinerecipients (>90%) and be capable of generating long term memoryresponses which will be required for memory recall and subsequentdisease protection post-infection. However, to generate this type ofdurable immunity a vaccine must also achieve a robust booster amplifyingeffect with repeat vaccine exposure.

Current immunological strategies to improve the cellular immunityinduced by vaccines and immunotherapeutics include the development oflive attenuated versions of the pathogen and the use of live vectors todeliver appropriate antigens or DNA coding for such antigens. Suchapproaches, which invariably fail to generate a meaningful boosterresponse in unselected populations, have led to convoluted prime-boostcombinations and are also limited by safety considerations within anincreasingly stringent regulatory environment. In addition, issuesarising from the scalability of manufacturing processes and prohibitivecosts often limit the commercial viability of products of biologicalorigin. In this context, synthetic peptides are very attractive antigensas they are chemically well-defined, highly stable and can be designedto contain T and/or B cell epitopes.

In order to stimulate T lymphocyte responses in vivo, synthetic peptidescontained in a vaccine or an immunotherapeutic product should preferablybe internalized by antigen presenting cells and especially dendriticcells. Dendritic cells (DCs) play a crucial role in the initiation ofprimary T-cell mediated immune responses. These cells exist in two majorstages of maturation associated with different functions. Immaturedendritic cells (iDCs) are located in most tissues or in the circulationand are recruited into inflamed sites in the body. They are highlyspecialised antigen-capturing cells, expressing large amounts ofreceptors involved in antigen uptake and phagocytosis. Following antigencapture and processing, iDCs move to local T-cell locations in the lymphnodes or spleen. During this process, DCs lose their antigen-capturingcapacity turning into immunostimulatory mature DCs (mDCs).

Dendritic cells are efficient presenting cells that initiate the host'simmune response to peptide antigen associated with class I and class IIMHC molecules. They are able to prime naïve CD4 and CD8 T-cells.According to current models of antigen processing and presentationpathways, exogeneous antigens are internalised into the endocyticcompartments of antigen presenting cells where they are degraded intopeptides, some of which bind to MHC class II molecules. The mature MHCclass II/peptide complexes are then transported to the cell surface forpresentation to CD4 T-lymphocytes. In contrast, endogenous antigen isdegraded in the cytoplasm by the action of the proteosome before beingtransported into the cytoplasm where they bind to nascent MHC class Imolecules. Stable MHC class I molecules complexed to peptides are thentransported to the cell surface to stimulate CD8 CTL. Exogenous antigenmay also be presented on MHC class I molecules by professional APCs in aprocess called cross-presentation. Phagosomes containing extracellularantigen may fuse with reticulum endoplasmic and antigen may gain themachinery necessary to load peptide onto MHC class I molecules

The Examples herein highlight the differential T-cell immune responseobtained by the attachment of a fluorocarbon vector to antigens comparedto the corresponding non-fluorinated antigens. The eight (8) antigensexemplified were selected from the list of Influenza sequences hereindefined. This provisional selection utilized a proprietary selectionalgorithm encompassing a combination of parameters including;immunoinformatics selection, in-vitro binding assays, ex-vivorestimulation assays using human PBMC previously infected withinfluenza, manufacturing and formulation parameters. Finally theassessment in mice confirmed that fluoropeptides thus selected eitherindividually or in combination were immunogenic and the responsesobtained were superior to the native peptide antigens. The antigenselection focus and desire to utilize a combination of antigens for thisvaccine prototype is such that both viral genetic and human HLAdiversity are addressed in this rational vaccine design. This has beenone of the key failings in the peptide vaccine field. Whilst it ispossible to utilize a single antigen in the fluoropeptide vaccine itwould limit the vaccine's immunogenicity potential in an outbred human(or other) population and therefore the selection of multiple peptidesis essential for a broadly effective vaccine.

As used herein (e.g., in the figures) the term “fluoropepetides” refersto fluorocarbon vectors (chains) conjugated to peptide based antigens.The Examples refer to the figures in which:

FIG. 1 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus its native peptide equivalent in BALB/c andCBF6 mice, after prime or prime-boost, assessed by ex vivo IFN-γ ELIspotassay. Seven or eight mice per group were immunized subcutaneously withthe fluoropeptide vaccine (composed of 8 formulated fluoropeptides at adose of 1 nmol per fluoropeptide in 100 μl) or the equivalent nativepeptides (composed of 8 formulated native peptides at a dose of 1 nmolper peptide in 100 μl). The control group received a formulationcontaining excipient only. Ten days after the final injection mice weresacrificed by cervical dislocation. Spleens were removed and singlespleen cell suspensions were prepared from individual mice. Murine IFN-γELISpot assays (Mabtech, Sweden) were performed according tomanufacturer's instructions. Spleen cells (5×10⁵) were stimulated, induplicate, with 8 individual native peptides at a concentration of 10μg/ml per peptide in complete culture medium (RPMI supplemented with 10%Foetal Calf Serum) in a total volume of 200 μl for 18 hours at 37° C.and 5% CO₂. The spots were counted using a CTL-immunospot reader unit.For each mouse, the ft) total number of spots was cumulated for all 8peptides and the value of the control wells (media only) was subtracted8 times. The results correspond to mean±standard deviation of spotforming cells (SFC) per million input spleen cells.

FIG. 2 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus its native peptide equivalent in BALB/c andCBF6 mice, after prime or prime-boost, assessed by ex vivo IFN-γ ELIspotassay. Seven or eight mice per group were immunized subcutaneously withthe fluoropeptide vaccine (composed of 8 formulated fluoropeptides at adose of 1 nmol per fluoropeptide in 100 μl) or the equivalent nativepeptides (composed of 8 formulated native peptides at a dose of 1 nmolper peptide in 100 μl). The control group received a formulationcontaining excipient only. Ten days after the final injection mice weresacrificed by cervical dislocation. Spleens were removed and singlespleen cell suspensions were prepared from individual mice. Murine IFN-γELISpot assays (Mabtech, Sweden) were performed according tomanufacturer's instructions. Spleen cells (5×10⁵) were stimulated, induplicate, with a mixture of 8 peptides at a concentration of 1 μg/mlper peptide in complete culture medium (RPMI supplemented with 10%Foetal Calf Serum) in a total volume of 200 μl for 18 hours at 37° C.and 5% CO₂. The spots were counted using a CTL-immunospot reader unit.For each mouse, the total number of spots was cumulated for all 8peptides and the value of the control wells (media only) was subtracted8 times. The results correspond to mean±standard deviation of spotforming cells (SFC) per million input spleen cells.

FIG. 3 shows a comparison of individual peptide immunogenicity offluoropeptides verses native peptides in BALB/c and CBF6 mice afterprime or prime-boost assessed by ex vivo IFN-γ ELISpot. Seven or eightmice per group were immunized subcutaneously with the fluoropeptidevaccine (composed of 8 formulated fluoropeptides at a dose of 1 nmol perfluoropeptide in 100 μl) or the equivalent native peptides (composed of8 formulated native peptides at a dose of 1 nmol per peptide in 100 μl).The control group received a formulation containing excipient only. Tendays after the last injection mice were sacrificed by cervicaldislocation. Spleens were removed and single spleen cell suspensionswere prepared from individual mice. Murine IFN-γ ELISpot assays(Mabtech, Sweden) were performed according to manufacturer'sinstructions. Spleen cells (5×10⁵) were stimulated, in duplicate, with 8individual native peptides at a concentration of 10 μg/ml per peptide incomplete culture medium (RPMI supplemented with 10% Foetal Calf Serum)in a total volume of 200 μl for 18 hours at 37° C. under 5% CO₂atmosphere. The spots were counted using a CTL-immunospot reader unit.The results correspond to mean±standard deviation of spot forming cells(SFC) per million input spleen cells.

FIG. 4 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus its native peptide equivalent in BALB/c andCBF6 mice after prime-boost immunization; assessment of cytokineprofiles. Eight mice per group were immunized subcutaneously with thefluoropeptide vaccine (composed of 8 formulated fluoropeptides at a doseof 1 nmol per fluoropeptide in 100 μl) or the equivalent native peptides(composed of 8 formulated native peptides at a dose of 1 nmol perpeptide in 100 μl). The control groups of mice were injected with aformulation containing excipient only. Mice were immunized at a 15 dayinterval. Ten days after the last injection mice were sacrificed bycervical dislocation. Spleens were removed and single spleen cellsuspensions were prepared from individual mice. Splenocytes werestimulated with a mixture of 8 native peptides at a concentration of 1μg/ml per peptide in complete culture medium (RPMI supplemented with 10%Foetal Calf Serum) in a total volume of 200 μl for 48 hours at 37° C.under 5% CO₂ atmosphere. Analysis of cytokine concentrations(interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5),interferon-γ (IFN-γ), and Tumor Necrosis Factor (TNF)) from the culturesupernatants of stimulated cells was conducted using a murine cytometricbead array kit (CBA; BD Biosciences, UK) according to manufacturer'sinstructions and was analyzed using a FacsCanto II flow cytometer.Standard curves were determined for each cytokine from a range of210-2500 pg/ml. The lower limit of detection for the CBA, according tothe manufacturer, is 2.5-3.2 pg/ml, depending on the analyte. Theresults correspond to mean values and standard deviation calculated foreach group of mice for each cytokine. Results are expressed as cytokineconcentration in pg/ml.

FIG. 5 is a graph showing that both CD4+ T cells and CD8+ T cells arestimulated by the fluoropeptide vaccine in BALB/c mice. Four mice pergroup were immunized subcutaneously with the fluoropeptide vaccine(composed of 8 formulated fluoropeptides at a dose of 1 nmol perfluoropeptide in 100 μl). Mice received 2 injections (prime-boost) at a15 day interval. Ten days after the last injection, mice were sacrificedby cervical dislocation. Spleens were removed and single spleen cellsuspensions were prepared from individual mice. Cells were resuspendedat 0.5×10⁶/well and stimulated with media only or a mixture of 8 nativepeptides (vaccine) for 72 hours at 37° C. and 5% CO₂. Positive controlcultures (PMA/I) received 50 ng/ml PMA and 0.5 μg/ml ionomycin for thefinal 5 hours of culture. All cultures received 10 μl/ml Brefeldin A forthe final 5 hours of culture. Cells were stained extracellularly for CD4and CD8, and intracellularly for IFN-γ, and analyzed by flow cytometryusing a BD FACSCanto II cytometer. Results for individual mice are shownas percentage of CD4+ or CD8+ T cells expressing intracellular IFN-γ.

FIG. 6 shows a comparison of the immunogenicity of a multivalentfluoropeptide vaccine versus vaccine emulsified in CFA in BALB/c miceafter a single immunization; assessment of cytokine profiles. Ten miceper group were immunized subcutaneously with the fluoropeptide vaccine(composed of 8 formulated fluoropeptides at a dose of 1 nmol perfluoropeptide in 100 μl) or fluoropeptide vaccine emulsified in completeFreund's adjuvant (CFA). The control group of mice was injected with aformulation containing excipient only. Ten days later mice weresacrificed by cervical dislocation. Spleens were removed and singlespleen cell suspensions were prepared from individual mice. Splenocyteswere stimulated with a mixture of 8 native peptides at a concentrationof 1 μg/ml per peptide in complete culture medium (RPMI supplementedwith 10% Foetal Calf Serum) in a total volume of 200 μl for 48 hours at37° C. under 5% CO₂ atmosphere. Analysis of cytokine concentrations(interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5),interferon-γ (IFN-γ), and Tumor Necrosis Factor (TNF)) from the culturesupernatants of stimulated cells was conducted using a murine cytometricbead array kit (CBA; BD Biosciences, UK) according to manufacturer'sinstructions and was analyzed using a FacsCanto II flow cytometer.Standard curves were determined for each cytokine from a range of2.5-2500 pg/ml. The lower limit of detection for the CBA, according tothe manufacturer, is 2.5-3.2 pg/ml, depending on the analyte. Theresults correspond to mean values±standard error calculated for eachgroup of mice for each cytokine. Results are expressed as mean cytokineconcentration in pg/ml.

FIG. 7 shows a comparison of subcutaneous versus intradermal routes offluoropeptide vaccine administration in BALB/c mice after a singleimmunization: ex vivo IFN-γ ELISpot assay. Ten mice per group wereimmunized subcutaneously (s.c.) or intradermally (i.d.) with thefluoropeptide vaccine (composed of 8 formulated fluoropeptides at a doseof 1 nmol per fluoropeptide in 100 μl). The control group received aformulation containing excipient only administered subcutaneously. Tendays later mice were sacrificed by cervical dislocation. Spleens wereremoved and single spleen cell suspensions were prepared from individualmice. Murine IFN-γ ELISpot assays (Mabtech, Sweden) were performedaccording to manufacturer's instructions. Spleen cells (5×10⁵) werestimulated, in duplicate, with 8 individual native peptides at aconcentration of 10 μg/ml per peptide in complete culture medium (RPMIsupplemented with 10% Foetal Calf Serum) in a total volume of 200 μl for18 hours at 37° C. under 5% CO₂ atmosphere. The spots were counted usinga CTL-immunospot reader unit. For each mouse, the total number of spotswas cumulated for all 8 peptides and the value of the control wells(media only) was subtracted 8 times. The results correspond tomean±standard error of spot forming cells (SFC) per million input spleencells.

In light of the foregoing description, the specific non-limitingexamples presented below are for illustrative purposes and not intendedto limit the scope of the invention in any way.

EXAMPLES Example 1 Example Peptides

Candidates for conjugating to a fluorocarbon vector for inclusion into aprophylactic or therapeutic vaccine for influenza may include thefollowing one or more peptides or fragments thereof, or homologues(including the corresponding consensus, ancestral or central treesequences as referred to in the Los Alamos National Laboratory influenzasequence database (Macken, C., Lu, H., Goodman, J., & Boykin, L., “Thevalue of a database in surveillance and vaccine selection.” in Optionsfor the Control of Influenza IV. A. D. M. E. Osterhaus, N. Cox & A. W.Hampson (Eds.) 2001, 103-106.) or Influenza virus resources at NCBI) ornatural and non-natural variants thereof, but not necessarilyexclusively. Specific examples of appropriate peptides are given belowwhere the standard one letter code has been utilized. Homologues have atleast a 50% identity compared to a reference sequence. Preferably ahomologue has 80, 85, 90, 95, 98 or 99% identity to a naturallyoccurring sequence. The use of non-natural amino acids must notinterfere with the ability of the peptide to bind to MHC class I or IIreceptors. Fragments of these sequences that contain one or moreepitopes are also candidate peptides for attachment to the fluorocarbonvector.

These sequences were selected from Influenza A consensus sequences. Theinfluenza virus protein and the position of the peptide within thatprotein are specified. Protein sequences were collected from theInfluenza virus resource web site, at ncbi.nlm.nih.gov/genomes/FLU/.

SEQ ID No1 PB2 Position 027 to 061HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADK (SEQ ID NO: 1) SEQ ID No2PB2 Position 123 to 157ERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAK (SEQ ID NO: 2) SEQ ID No3PB2 Position 155 to 189SAKEAQDVIMEVVFPNEVGARILTSESQLTITKEK (SEQ ID NO: 3) SEQ ID No4PB2 Position 203 to 237VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQG (SEQ ID NO: 4) SEQ ID No5PB2 Position 249 to 283EVRNDDVDQSLIIAARNIVRRAAVSADPLASLLEM (SEQ ID NO: 5) SEQ ID No6PB2 Position 358 to 392EGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQ (SEQ ID NO: 6) SEQ ID No7PB2 Position 370 to 404ATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVF (SEQ ID NO: 7) SEQ ID No8PB2 Position 415 to 449RGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQNW (SEQ ID NO: 8) SEQ ID No9PB2 Position 532 to 566SSSMMWEINGPESVLVNTYQWIIRNWETVKIQWSQ (SEQ ID NO: 9) SEQ ID No10PB2 Position 592 to 626 YSGFVRTLFQQMRDVLGTFDTVQIIKLLPFAAAPP (SEQ ID NO:10) SEQ ID No11 PB2 Position 607 to 641LGTFDTVQIIKLLPFAAAPPEQSRMQFSSLTVNVR (SEQ ID NO: 11) SEQ ID No12PB2 Position 627 to 659QSRMQFSSLTVNVRGSGMRILVRGNSPVFNYNK (SEQ ID NO: 12) SEQ ID No13PB1 Position 012 to 046 VPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRT (SEQ ID NO:13) SEQ ID No14 PB1 Position 114 to 148VQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIE (SEQ ID NO: 14) SEQ ID No15PB1 Position 216 to 250 SYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRG (SEQ ID NO:15) SEQ ID No16 PB1 Position 267 to 301EQSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFT (SEQ ID NO: 16) SEQ ID No17PB1 Position 324 to 358 YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE (SEQ ID NO:17) SEQ ID No18 PB1 Position 340 to 374APIMFSNKMARLGKGYMFESKXMKLRTQIPAEMLA (SEQ ID NO: 18) SEQ ID No19PB1 Position 404 to 436SPGMMMGMFNMLSTVLGVSILNLGQKKYTKTTY (SEQ ID NO: 19) SEQ ID No20PB1 Position 479 to 513 KKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFG (SEQ ID NO:20) SEQ ID No21 PB1 Position 486 to 520KTGTFEFTSFFYRYGFVANFSMELPSFGVSGINES (SEQ ID NO: 21) SEQ ID No22PB1 Position 526 to 560 GVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR (SEQ ID NO:22) SEQ ID No23 PB1 Position 656 to 690EYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQ (SEQ ID NO: 23) SEQ ID No24PB1 Position 700 to 734 FPSSSYRRPVGISSMVEAMVSRARIDARIDFESGR (SEQ ID NO:24) SEQ ID No25 PA Position 107 to 141PDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSE (SEQ ID NO: 25) SEQ ID No26PA Position 122 to 156 VTRREVHIYYLEKANKIKSEKTHIHIFSFTGEEMA (SEQ ID NO:26) SEQ ID No27 PA Position 145 to 179IHIFSFTGEEMATKADYTLDEESRARIKTRLFTIR (SEQ ID NO: 27) SEQ ID No28PA Position 166 to 200 ESRARIKTRLFTIRQEMASRGLWDSFRQSERGEET (SEQ ID NO:28) SEQ ID No29 PA Position 495 to 529RRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTD (SEQ ID NO: 29) SEQ ID No30PA Position 642 to 676 AKSVFNSLYASPQLEGFSAESRKLLLIVQALRDNL (SEQ ID NO:30) SEQ ID No31 PA Position 173 to 207PRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFW (SEQ ID NO: 31) SEQ ID No32NP Position 240 to 274 DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS (SEQ ID NO:32) SEQ ID No33 M1 Position 002 to 026SLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKN (SEQ ID NO: 33) SEQ ID No34M1 Position 023 to 057 EIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTK (SEQ ID NO:34) SEQ ID No35 M1 Position 038 to 072DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER (SEQ ID NO: 35) SEQ ID No36M1 Position 055 to 089 LTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGD (SEQ ID NO:36) SEQ ID No37 M1 Position 166 to 200ATTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAA (SEQ ID NO: 37) SEQ ID No38NS1 Position 128 to 162 IILKANFSVIFDRLETLILLRAFTEEGAIVGEISP (SEQ ID NO:38) SEQ ID No39 NS2 Position 026 to 060EDLNGMITQFESLKLYRDSLGEAVMRMGDLHSLQN (SEQ ID NO: 39)

The following sequences were selected from Influenza B consensussequences. The influenza virus protein and the position of the peptidewithin that protein are specified. Protein sequences were collected fromthe Influenza virus resource web site, at ncbi.nlm.nih.gov/genomes/FLU/:

SEQ ID No40 PB2 Position 016 to 050NEAKTVLKQTTVDQYNIIRKFNTSRIEKNPSLRMK (SEQ ID NO: 40) SEQ ID No41PB2 Position 117 to 151 YESFFLRKMRLDNATWGRITFGPVERVRKRVLLNP (SEQ ID NO:41) SEQ ID No42 PB2 Position 141 to 175ERVRKRVLLNPLTKEMPPDEASNVIMEILFPKEAG (SEQ ID NO: 42) SEQ ID No43PB2 Position 197 to 231 GTMITPIVLAYMLERELVARRRFLPVAGATSAEFI (SEQ ID NO:43) SEQ ID No44 PB2 Position 311 to 345DIIRAALGLKIRQRQRFGRLELKRISGRGFKNDEE (SEQ ID NO: 44) SEQ ID No45PB2 Position 404 to 438 MVFSQDTRMFQGVRGEINFLNRAGQLLSPMYQLQR (SEQ ID NO:45) SEQ ID No46 PB2 Position 519 to 553VSELESQAQLMITYDTPKMWEMGTTKELVQNTYQW (SEQ ID NO: 46) SEQ ID No47PB2 Position 537 to 571 MWEMGTTKELVQNTYQWVLKNLVTLKAQFLLGKED (SEQ ID NO:47) SEQ ID No48 PB2 Position 572 to 606MFQWDAFEAFESIIPQKMAGQYSGFARAVLKQMRD (SEQ ID NO: 48) SEQ ID No49PB2 Position 717 to 751 LEKLKPGEKANILLYQGKPVKVVKRKRYSALSNDI (SEQ ID NO:49) SEQ ID No50 PB1 Position 001 to 035MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTG (SEQ ID NO: 50) SEQ ID No51PB1 Position 097 to 131 EEHPGLFQAASQNAMEALMVTTVDKLTQGRQTFDW (SEQ ID NO:51) SEQ ID No52 PB1 Position 227 to 261MTKDAERGKLKRRAIATAGIQIRGFVLVVENLAKN (SEQ ID NO: 52) SEQ ID No53PB1 Position 393 to 427 KPFFNEEGTASLSPGMMMGMFNMLSTVLGVAALGI (SEQ ID NO:53) SEQ ID No54 PB1 Position 616 to 650DPEYKGRLLHPQNPFVGHLSIEGIKEADITPAHGP (SEQ ID NO: 54) SEQ ID No55PB1 Position 701 to 735 SASYRKPVGQHSMLEAMAHRLRMDARLDYESGRMS (SEQ ID NO:55) SEQ ID No56 PA Position 160 to 194SSLDEEGKGRVLSRLTELQAELSLKNLWQVLIGEE (SEQ ID NO: 56) SEQ ID No57PA Position 491 to 525 ESFDMLYGLAVKGQSHLRGDTDVVTVVTFEFSSTD (SEQ ID NO:57) SEQ ID No58 PA Position 696 to 723VIQSAYWFNEWLGFEKEGSKVLESVDEIMDE (SEQ ID NO: 58) SEQ ID No59NP Position 173 to 207 FLKEEVKTMYKTTMGSDGFSGLNHIMIGHSQMNDV (SEQ ID NO:59) SEQ ID No60 NP Position 253 to 287EAIRFIGRAMADRGLLRDIKAKTAYEKILLNLKNK (SEQ ID NO: 60) SEQ ID No61NP Position 308 to 342 IADIEDLTLLARSMVVVRPSVASKVVLPISIYAKI (SEQ ID NO:61) SEQ ID No62 NP Position 338 to 372IYAKIPQLGFNVEEYSMVGYEAMALYNMATPVSIL (SEQ ID NO: 62) SEQ ID No63NP Position 418 to 452 GFHVPAKEQVEGMGAALMSIKLQFWAPMTRSGGNE (SEQ ID NO:63) SEQ ID No64 M1 Position 166 to 300ARSSVPGVRREMQMVSAMNTAKTMNGMGKGEDVQK (SEQ ID NO: 64) SEQ ID No65M1 Position 209 to 237 IGVLRSLGASQKNGEGIAKDVMEVLKQSS (SEQ ID NO: 65)

Candidate peptides for inclusion into a prophylactic or therapeuticvaccine for influenza may be peptides from any of the viral proteinshaemagglutinin, neuraminidase, matrix (M1) protein, M2, nucleoprotein(NP), PA, PB1, PB2, NS1 or NS2 in any such combination.

Synthesis of Fluoropeptides and Native Peptides (Unmodified Peptides)

Eight native peptides and 8 fluoropeptides (selected from the peptidelist contained herein; SEQ ID No 1 through 65) were obtained by solidphase peptide synthesis (SPPS). All peptides were synthesized on Rinkamide PEG resin by using standard 9-fluorenyhnethoxycarbonyl (Fmoc)chemistry. The peptide chain was assembled on resin by repetitiveremoval of the Fmoc protecting group by treating with 20%piperidine/N,N-Dimethylformamide for 30 minutes and coupling ofprotected amino acid by using1,3-diisopropylcarbodiimide/1-hydroxy-benzotriazole/N-methylmorpholinefor 120 minutes. Ninhydrin test was performed after each coupling tocheck the coupling efficiency. After the addition of the N-terminalLysinyl residue, the resin blocks were split to allow (1) on the firsthalf of the resin, the incorporation of the2H,2H,3H,3H-Perfluoroundecanoic acid fluorocarbon chain(C₈F₁₇(CH₂)₂COOH) on the Epsilon-chain of the N-terminal lysine toderive the fluoropeptide and (2) on the second half of the resin, theacetylation of the Epsilon-chain of the N-terminal lysine to derive thenative peptide. Resins were washed and dried, then treated with reagentK for cleavage and removal of the side chain protecting groups. Crudepeptides were precipitated from cold ether and collected by filtration.Purity was assessed by RP-HPLC and was superior to 92% for all peptides.Freeze-dried fluoropeptides were prepared under nitrogen and stored at−20° C. Stability of the fluoropeptides under storage conditions havebeen confirmed by RP-HPLC and LC-MS over 6 months.

Vaccine Dose Preparation

Eight freeze-dried fluoropeptides (fluoropeptide 1, fluoropeptide 2,fluoropeptide 3, fluoropeptide 4, fluoropeptide 5, fluoropeptide 6,fluoropeptide 7 & fluoropeptide 8) or eight freeze-dried equivalentnative peptides (peptide 1, peptide 2, peptide 3, peptide 4, peptide 5,peptide 6, peptide 7 & peptide 8) were formulated to create an isomolarformulation yielding a broadly neutral pH for parenteral delivery.

The sequences of the influenza peptide portions of the constructs wereas follows (shown with an NH₂ cap on the carboxy terminus):

Fluoropeptide 1 (SEQ ID NO: 1) HMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADK-NH₂Fluoropeptide 2 (SEQ ID NO: 4) VAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQG-NH2Fluoropeptide 3 (SEQ ID NO: 17) YITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFE-NH2Fluoropeptide 4 (SEQ ID NO: 18) APIMFSNKMARLGKGYMFESKXMKLRTQIPAEMLA-NH2Fluoropeptide 5 (SEQ ID NO: 19) SPGMMMGMFNMLSTVLGVSILNLGQKKYTKTTY-NH2Fluoropeptide 6 (SEQ ID NO: 20) KKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFG-NH2Fluoropeptide 7 (SEQ ID NO: 32) DQVRESRNPGNAEIEDLIFLARSALILRGSVAHKS-NH2Fluoropeptide 8 (SEQ ID NO: 35) DLEALMEWLKTRPILSPLTKGILGFVFTLTVPSER-NH2

Animals and Immunization

Female, 6-8 weeks of age, BALB/c or CB6F1 (BALB/c×C57BL/6J) mice werepurchased from Charles River (UK) &/or Harlan (UK). Injections wereperformed subcutaneously using 1 ml syringe and 22-G needle.Immunizations were performed so that mice received either a singleimmunization (prime) or two immunizations (prime/boost). Immunizationswere performed with a 14 day interval between each injection.

Fluoropeptide Vaccine is Strongly Immunogenic and is Superior to NativePeptides in Both BALB/c and CB6F1 Mice

The immunogenicity of the fluoropeptide vaccine (mixture of 8fluoropeptides as above) was compared to the native peptide equivalent(mixture of 8 unmodified peptides—called native peptides as above) inBALB/c and CB6F1 mice. The study also compared the immunogenicity ofboth formulations using a prime or prime-boost regimen. Bothformulations were injected subcutaneously without adjuvant in BALB/c andCBF6 mice. Mice were immunized with a fluoropeptide vaccine dosecontaining 1 nmol/fluoropeptide (8 nmol total for eight fluoropeptides)or the native peptide vaccine equivalent at 1 nmol/peptide (8 nmol totalfor eight native peptides). Neither vaccine preparation contained anyadjuvant. 10 days after the final immunization, spleen cells wererestimulated with each individual native peptide at 10 μg/ml andassessed using an IFN-γ ELISpot assay. According to ex vivo IFN-γELISpot assays (FIGS. 1 & 2), the immunogenicity of the fluoropeptidevaccine was superior to both the excipient alone and the native peptidevaccine equivalent after a prime-boost immunization regimen (P<0.001).The results also demonstrated a strong increase in the number of spotforming cells using a prime-boost regimen compared to a singleimmunization for the fluoropeptide vaccine group only (FIGS. 1 & 2).These results demonstrate the self-adjuvanticity property of thefluorocarbon chain linked to a peptide sequences.

Fluoropeptide Vaccine Induces a Robust Multiepitopic T Cell Response inBoth BALB/c and CB6F1 Mice

The immunogenicity of the fluoropeptide vaccine (mixture of 8fluoropeptides as above) was compared to its native peptide equivalent(mixture of 8 unmodified peptides—referred to as ‘native peptides’ asabove) in BALB/c and CB6F1 mice. The study also compared theimmunogenicity of both formulations on a prime and prime-boost regimen.Both formulations were injected subcutaneously without adjuvant inBALB/c and CB6F1 mice. Mice were immunized with a fluoropeptide vaccinedose containing 1 nmol/fluoropeptide (8 nmol total for eightfluoropeptides), the native peptide vaccine equivalent at 1 nmol/peptide(8 nmol total for eight native peptides). Neither vaccine preparationcontained any adjuvant. The control group consisted of mice immunizedwith excipient alone. 10 days after immunization, spleen cells wererestimulated by each individual native peptide at 10 μg/ml and assessedusing IFN-γ ELISpot assay. The fluoropeptide vaccine inducepeptide-specific responses directed against 5 out of 8 peptides inBALB/c mice and 7 out of 8 peptides in CB6F1 mice which is superior tothe response induced by the vaccine equivalent (unmodified peptides).This demonstrates that vaccination with fluoropeptides can induce animmunological response that is both qualitatively and quantitativelysuperior to that of its native peptide equivalent.

The Fluoropeptide Vaccine Induces a Th1 Cytokine Profile Depending Uponthe Murine Strain Tested

The immunogenicity of the fluoropeptide vaccine (mixture of 8fluoropeptides as above) was compared to the native peptide equivalent(mixture of 8 unmodified peptides as above) in BALB/c and CB6F1 mice.Formulations were injected subcutaneously without adjuvant in BALB/c andCB6F1 mice. Mice were immunized with a fluoropeptide vaccine dosecontaining 1 nmol/fluoropeptide (8 nmol total for eight fluoropeptides),the native peptide vaccine equivalent at 1 nmol/peptide (8 nmol totalfor eight native peptides). Neither vaccine preparation contained anyadjuvant. 10 days after the last immunization, spleen cells wererestimulated with a mixture of 8 native peptides at 1 μg/ml per peptide.After 48 hours stimulation culture supernatants were assessed forcytokines by means of a multiplexed bead assay (CBA). Resultsdemonstrate the cytokine profile in CBF6 mice is dominated by theproduction of IFN-γ and significant production of TNF-α highlighting aTh1 profile (FIG. 4). This Th1-dominated cytokine profile was morepronounced compared to BALB/c mice due to a lower intensity of these Th1responses compared to CB6F1 mice (as also observed by IFN-γELISpot—refer to FIGS. 1 & 2) and increases in Th2 cytokines.Nevertheless, an enhanced Th1 response was observed in BALB/c miceimmunized with fluoropeptides compared to its native peptide equivalent.

The Fluoropeptide Vaccine Stimulates Both Peptide-Specific CD4+ and CD8+T Cells Producing IFN-γ

Intracellular cytokine staining for IFN-γ was used to provideinformation about the frequency of peptide-specific CD4+ and CD8+ Tcells producing IFN-γ. Mice were immunized with the fluoropeptidevaccine (mixture of 8 fluoropeptides as above) and CD4+ or CD8+splenocytes were assessed for intracellular cytokine staining by flowcytometry after a short stimulation period with a mixture of 8 nativepeptides (vaccine). The results demonstrate that immunization of micewith the fluoropeptide vaccine was able to elicit both peptide-specificCD4+ and CD8+ T cells producing IFN-γ at a frequency of 0.5-2.6% (FIG.5). This validates that fluoropeptides engage both MHC class I & IIantigen processing peptides if the peptides contain relevant MHC class I& II epitopes.

Example 2 Immune Responses Elicited by Fluoropeptide Vaccination areBoosted by Combination with Adjuvant

Immunogenicity of the fluoropeptide vaccine (mixture of 8 fluoropeptidesas above) was compared with immunogenicity of the fluoropeptide vaccinein the presence of an adjuvant, Freund's complete adjuvant (FCA).Fluoropeptide vaccine (1 nmol/peptide) or fluoropeptide vaccine (1nmol/peptide) emulsified in CFA was used to immunize BALB/c mice. 10days after the immunization, splenocytes were stimulated with individualpeptides at 10 μg/ml. 48 hours later culture supernatants were collectedand tested for cytokines using a multiplex cytokine assay (CBA). Resultsshow that using an CFA as an additional adjuvant can significantly boostTh1 cytokine production (IFN-γ and IL-2) without effecting theproduction of Th2 cytokines (IL-4, IL-5) (FIG. 6). Therefore Th1responses induced by fluoropeptide vaccination are preferentiallyboosted by combination with adjuvant during immunization.

Both Subcutaneous and Intradermal Routes of Fluoropeptide VaccineAdministration can Induce Immune Responses

Immunogenicity of the fluoropeptide vaccine (mixture of 8 fluoropeptidesas above) was compared using either intradermal or subcutaneous routesof administration in BALB/c mice. 10 days after the immunization,splenocytes were stimulated with individual peptides at 10 μg/ml andassessed for ex vivo IFN-γ production by means of ELISPOT. Results showthat both subcutaneous and intradermal routes of fluoropeptideadministration are suitable to induce robust antigen-specific responses(FIG. 7).

INCORPORATION BY REFERENCE

The entire disclosure of each of the publications and patent documentsreferred to herein is incorporated by reference in its entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-33. (canceled)
 34. A vector-antigen construct comprising: an antigenicinfluenza peptide sequence covalently attached to a vector construct,wherein the vector construct comprises a chain comprising from 3 to 30carbon atoms at least one of which is substituted with a fluorine,chlorine, bromine or iodine and wherein the antigenic influenza peptideis 15 to 40 amino acids in length and comprises at least one MHC Iepitope and at least one MHC II epitope.
 35. The vector-antigenconstruct of claim 34, wherein the antigenic influenza peptide comprisesthe amino acid sequence of any one of SEQ ID NOs: 1 to
 65. 36. Thevector-antigen construct of claim 34, wherein the antigenic influenzapeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1,4, 17, 18, 32 or
 35. 37. The vector-antigen construct of claim 34,wherein the vector is of structure C_(m)F_(n)—C_(y)H_(x)-(Sp)-R, whereinm=3 to 30, n<=2m+1, y=0 to 15, x<=2y, (m+y)=3 to 30, Sp is an optionalchemical spacer moiety and R is the antigenic influenza peptide.
 38. Thevector-antigen construct of claim 34, wherein the vector comprisesstructure

where Sp is an optional chemical spacer moiety and R is the antigenicinfluenza peptide.
 39. A pharmaceutical composition for intracellulardelivery of an antigen, the composition comprising an antigenicinfluenza peptide sequence covalently attached to a vector construct,wherein the vector construct comprises a chain comprising from 3 to 30carbon atoms at least one of which is substituted with a fluorine,chlorine, bromine or iodine and wherein the antigenic influenza peptideis 15 to 40 amino acids in length and comprises at least one MHC Iepitope and at least one MHC II epitope.
 40. The composition of claim39, wherein the antigenic influenza peptide comprises the amino acidsequence of any one of SEQ ID NOs: 1 to
 65. 41. The composition of claim39, wherein the vector is of structure C_(m)F_(n)—C_(y)H_(x)-(Sp)-R,wherein m=3 to 30, n<=2m+1, y=0 to 15, x<=2y, (m+y)=3 to 30, Sp is anoptional chemical spacer moiety and R is the antigenic influenzapeptide.
 42. The composition of claim 39, wherein the vector comprisesstructure

where Sp is an optional chemical spacer moiety and R is the antigenicinfluenza peptide.
 43. The composition of claim 39, wherein thecomposition comprises from 2 to 20 of the antigenic influenza peptidesequences each covalently attached to a vector construct.
 44. Thecomposition of claim 39, wherein the composition comprises 5, 6, 7, or 8of the antigenic influenza peptide sequences each covalently attached toa vector construct.
 45. The composition of claim 39, further comprisingone or more pharmaceutically acceptable carriers, excipients, diluentsor adjuvants.
 46. The composition of claim 39, formulated forparenteral, oral, ocular, rectal, nasal, transdermal, topical or vaginaladministration.
 47. The composition of claim 39, wherein the compositionis in the form of a liquid, emulsion, solid, aerosol or gas.
 48. Amethod of stimulating an immune response, comprising administering thecomposition of claim 39 to an animal.
 49. The method of claim 48,wherein the animal is a mammal, a bird or a human.
 50. A method ofpreparing a prophylactic or therapeutic pharmaceutical compositioncomprising combining the composition of claim 39, with one or morepharmaceutically acceptable carriers, excipients, diluents or adjuvants.